Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct [alpha] chains. We investigated the cellular regulation and origin of these [alpha](IV) chains in normal and neoplastic breast tissues by immunohistochemistry by using [alpha](IV) chain-specific antibodies and by in situ hybridization. In normal breast, [alpha]1(IV) and [alpha]2(IV) chains were stained in all BM, whereas [alpha]5(IV) and [alpha]6(IV) chains were restrictively localized in a linear pattern in the BM of the mammary gland. Similar immunostaining profiles were observed in benign breast tumors and in the intraductal components of invasive ductal carcinoma. However, in invasive ductal carcinoma, [alpha]1(IV) and [alpha]2(IV) chains were discontinuously or negatively stained in the cancer cell nests, and the assembly of [alpha]5(IV) and [alpha]6(IV) chains into the BM was completely inhibited. Coexpression of [alpha]5(IV) and [alpha]6(IV) chains was related to the localization of [alpha]-smooth muscle actin ([alpha]-SMA)-positive myoepithelial cells. By in situ hybridization, in fibroadenoma and invasive ductal carcinoma, the signals for [alpha]1(IV) and [alpha]2(IV) mRNA were abundant in stromal cells. However, the signals for [alpha]5(IV) and [alpha]6(IV) mRNA were not seen in any of these cells. In contrast, in intraductal papilloma, coexpression of [alpha]1(IV)/[alpha]2(IV) mRNA and [alpha]5(IV)/[alpha]6(IV) mRNA was identified in epithelial cells. The results indicate that the mammary gland forms a second network of BM composed of [alpha]5(IV)/[alpha]6(IV) chains, in addition to the classic network of [alpha]1(IV)/[alpha]2(IV) chains. The expression of type IV collagen [alpha] chains seems to be differentially regulated by the epithelial-myoepithelial interaction and to be associated with the invasive potential of breast cancer. Lab Invest 1999, 79:281-292).